While powerful, phenotype driven genetic screens in mammalian cells are limited by the diploid state of their genome and the inability to perform genetic crosses. Genetic screens reliant on RNA interference (RNAi) are commonly thought to surpass these limitations. This supposition is called into question however, when one considers problems inherently associated with gene silencing by RNAi, such as frequently observed off-target effects and the failure to completely silence gene expression. Genome-wide screening efforts, especially, may in fact founder from the accumulation of these insurmountable complications. Here we propose to bypass or alleviate such complications using a novel system allowing efficient genetic screening based on a haploid genomic context. The objective of this project is to develop a powerful new method for mammalian cell genetics for use in the identification of undiscovered genetic networks in cancer and other diseases. The specific aims are: 1. To generate a new platform for loss-of-function genetics. We will use a cell line haploid for all but one human chromosome and efficient gene-trap mutagenesis as an alternative methodology for phenotype-based, recessive genetic screens. 2. To use gene-trap screens in haploid cells to identify cancer relevant genes. We will employ this unique technology to discover novel genetic networks operative in cancer cell biology;our initial efforts aim to elucidate unknown genetic components of death-receptor induced apoptosis and therapeutic response to Gleevec. PUBLIC HEALTH RELEVANCE: Unbiased genetic screens are powerful means for elucidating poorly understood biological processes. RNA interference strategies comprise the most recent and widely used application for genetics in cultured mammalian cells. These approaches are undermined by the inherent rate of false positives and negatives. Here we propose to develop a new system for mammalian cell genetics using a cell system that is haploid for nearly all human chromosomes. We will further validate the utility of this approach by identifying novel, cancer-relevant genetic pathways. Following its development and validation, we foresee this novel strategy for mammalian genetic screens would be pertinently useful in more broad applications of biomedical research.